The structure and organization of the human bilirubin UDP- glucuronosyltransferase gene was determined by isolating 3 overlapping clones from a human lymphocyte DNA cosmid library which was screened with bilirubin transferase-specific cDNA probes previously isolated in our laboratory. Two areas of investigation were pursued. A. The UGT1 Locus. We showed that one phenol transferase and two bilirubin isoforms are encoded by an unusual gene locus (spanning about 95 kb), UGT1, which is also predicted to encode three other bilirubin transferase-like isozymes all having identical carboxyl termini. The transcriptional arrangement utilizes 6 nested promoter elements each of which is positioned upstream of a unique exon 1. Each exon 1 encodes the NH2-terminal domain and confers substrate specificity to the isoform. The 3' end of the locus contains 4 common exons which encode the identical carboxyl termini. It is predicted that 6 nested primary transcripts are synthesized and that each exon 1 is differentially spliced to the common exons to produce 6 unique, mature mRNAs. Expression of mRNA is tissue- specific and presumably differentially responsive to inducers and developmentally regulated. B. Preliminary Studies on Genetic Defects. In one Crigler-Najjar Type I patient we found evidence for a point mutation in the common region of the UGT1 locus. In a second patient, we found a 13-nucleotide deletion in the same general region of the locus. The importance of determining the structure of the human bilirubin transferase gene relates to the occurrence in the population of heritable lethal and nonlethal mutations within this enzyme system. Bilirubin is a potent neurotoxin produced from the normal turnover of red blood cells and is completely dependent upon bilirubin transferase for its detoxification.